© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1978-1983 doi:10.1242/dev.122325
RESEARCH REPORT
DEFECTIVE KERNEL 1 promotes and maintains plant epidermal
differentiation
Roberta Galletti1, *, Kim L. Johnson2, Simon Scofield3, Rita San-Bento1, Andrea M. Watt2, James A. H. Murray3
and Gwyneth C. Ingram1, *
During plant epidermal development, many cell types are generated
from protodermal cells, a process requiring complex co-ordination of cell
division, growth, endoreduplication and the acquisition of differentiated
cellular morphologies. Here we show that the Arabidopsis phytocalpain
DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal
state. Plants with reduced DEK1 activity produce cotyledon epidermis
with protodermal characteristics, despite showing normal growth and
endoreduplication. Furthermore, in non-embryonic tissues (true leaves,
sepals), DEK1 is required for epidermis differentiation maintenance. We
show that the HD-ZIP IV family of epidermis-specific differentiationpromoting transcription factors are key, albeit indirect, targets of DEK1
activity. We propose a model in which DEK1 influences HD-ZIP IV gene
expression, and thus epidermis differentiation, by promoting cell
adhesion and communication in the epidermis.
KEY WORDS: DEK1, Epidermis, Differentiation maintenance,
Arabidopsis thaliana
INTRODUCTION
The production of specific function-adapted organ morphologies in
plants is achieved by the exquisitely complex spatial and temporal
control of cell proliferation, expansion and differentiation. The
importance of this co-ordination is well illustrated in the cotyledon
epidermis of Arabidopsis thaliana. To perform its basic functions
this cell layer must produce both mature pavement cells (PCs),
which can be very large and have a complex morphology, and
appropriately spaced pairs of stomatal guard cells, which are tiny
and adapted to their role in gas exchange (Glover, 2000). Producing
this complex mosaic demands that neighbouring cells co-ordinate
their growth.
Epidermal specification and differentiation are linked to the activity
of subfamily IV of the plant-specific homeodomain-leucine zipper
(HD-ZIP) transcription factors (TFs) (Abe et al., 2003; DepegeFargeix et al., 2011; Di Cristina et al., 1996; Horstman et al., 2015;
Javelle et al., 2011a, 2010; Nakamura et al., 2006; Peterson et al.,
2013; Roeder et al., 2012; San-Bento et al., 2014; Takada et al., 2013;
Vernoud et al., 2009; Wu et al., 2011; Yang et al., 2011). Cell-cell
communication influences both epidermal cell fate decisions and
subsequent differentiation (reviewed by Javelle et al., 2011b;
1
Laboratoire de Reproduction et Dé veloppement des Plantes, Ecole Normale
2
Supé rieure de Lyon, 46 allé e d’Italie, Lyon 69364, Cedex 07, France. ARC Centre
of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Royal
3
Parade, Parkville, Victoria 3010, Australia. Cardiff School of Biosciences, Cardiff
University, Museum Avenue, Cardiff CF10 3AX, UK.
*Authors for correspondence ([email protected];
[email protected])
Received 19 January 2015; Accepted 7 April 2015
1978
Schiefelbein et al., 2014; Takada and Iida, 2014). For example,
several receptor-like kinases (RLKs) and ligand-like peptides control
patterning of the stomatal lineage (reviewed by Richardson and Torii,
2013) and the CRINKLY4 (CR4) RLK, and its Arabidopsis
homologue ACR4, are required for the control of epidermal identity
in maize (Becraft et al., 1996) and the expression of HD-ZIP IV genes
in Arabidopsis (San-Bento et al., 2014).
DEFECTIVE KERNEL 1 (DEK1) regulates growth coordination
and epidermal characters in plants (Becraft et al., 2002; Johnson et al.,
2005, 2008; Lid et al., 2002). Loss of DEK1 function causes early
embryo lethality in both Arabidopsis and maize (Becraft et al.,
2002; Johnson et al., 2005). In Petunia and Arabidopsis, strong
downregulation of DEK1 is accompanied by proliferation of
disorganised callus-like cells (Ahn et al., 2004; Lid et al., 2005).
DEK1 is also required for multicellular development in the moss
Physcomitrella patens (Demko et al., 2014; Liang et al., 2013; Perroud
et al., 2014). DEK1 has a modular structure, with several predicted
transmembrane domains, a linker domain and a cytoplasmically
localised C-terminal domain comprising a sequence similar to animal
calpains and a C2-like domain (Lid et al., 2002). The complete
C-terminal domain (which we refer to as the CALPAIN domain) is
sufficient to complement dek1 loss-of-function alleles, suggesting that
this is the active domain of the protein (Johnson et al., 2008; Liang
et al., 2013). A weak DEK1 allele, dek1-4, which causes a single amino
acid substitution in the C2-like domain, and mutant alleles of ACR4 and
the HD-ZIP IV-encoding genes HDG11 and ATML1, were recently
reported to lack sepal giant cells (GCs) (Roeder et al., 2012).
Here we aim to understand the role of DEK1 in epidermis
differentiation, and particularly to clarify its relationship with the
HD-ZIP IV TFs.
RESULTS AND DISCUSSION
dek1-4 shows protodermal characteristics in cotyledon
epidermis but no significant defects in cell expansion, ploidy
or cell cycle-related gene expression
We found that the PCs in the adaxial epidermis of dek1-4 were less
complex and more homogeneous than in wild-type (WT; Col-0)
controls (Fig. 1). Three-day-old Col-0 cotyledon PCs included small
cells with almost no lobes, intermediate cells with few lobes and
larger, fully interdigitated cells (Fig. 1A). At the same stage, dek1-4
PCs ranged from showing no lobes (polygonal shape) to showing
some interdigitation, although less than in Col-0 (Fig. 1B,C). We
noted an abnormally high frequency of four-way, or near four-way,
junctions (usually found in meristematic cells) in dek1-4 (Fig. 1B,C).
In the dek1-4 mutant, intermediate size and large cells were rounder
than WT cells of similar size (Fig. 1D). Despite their shape differences,
the average final cell size of PCs in dek1-4 and WT was similar
(Fig. 1E). Thus, PCs in dek1-4 have, on average, less complex shapes.
This cellular phenotype persisted in fully expanded cotyledons
(Fig. 1H,I). Neighbouring PCs in dek1-4 frequently had shared
DEVELOPMENT
ABSTRACT
RESEARCH REPORT
Development (2015) 142, 1978-1983 doi:10.1242/dev.122325
Fig. 1. DEK1 promotes cell differentiation. (A-C) Confocal
images of the adaxial side of 3-day-old Col-0 (A) and dek1-4
(B,C) Arabidopsis cotyledons stained with PI. Scale bars:
50 μm. (D) Epidermal cell area plotted against shape factor
(combined data from cells measured in four independent
seedlings). Guard cells were eliminated from the analysis.
n=135 for Col-0 and n=189 for dek1-4. (E) Average area±s.e. of
epidermal pavement cells (PCs). (F-I) Images and agarose
imprints of the adaxial side of 8-day-old Col-0 (F,H) and dek1-4
(G,I) seedlings. Asterisks indicate four-way junctions (B,C) or
neighbouring PCs with shared straight cell walls (I) in the
mutant. (J) Average area±s.e. of 8-day-old cotyledons. n=19
for Col-0 and n=16 for dek1-4. (K) Nuclear DNA content/ploidy
distribution profile of 10-day-old Col-0 and dek1-4 cotyledons.
n=nuclei counted per sample.
seedlings (supplementary material Fig. S1). Therefore, the
phenotypes observed are not associated with ploidy defects.
DEK1 controls epidermal differentiation via a pathway
involving multiple HD-ZIP IV-encoding genes
dek1-4 leaves showed a significantly higher proportion of trichomes
with more than three branches than WT controls (Wilcoxon rank
sum test, P=1.7–20) (Fig. 2A-C). A similar phenotype was described
for hdg11 and hdg12 hdg11 mutants (Nakamura et al., 2006).
We analysed the expression of HDG11, HDG12 and of additional
HD-ZIP IV genes (ATML1, PDF2, HDG2) in the dek1-4 mutant and
found that all genes tested were reproducibly downregulated
(Fig. 2D). We then assessed the genetic relationship between
DEK1, HDG11 and HDG12. hdg12-2 dek1-4 double mutants
showed highly branched trichomes, as seen in the dek1-4 parental
1979
DEVELOPMENT
straight walls (Fig. 1I) suggesting that they had undergone late
divisions (observed in 100% of mutant seedlings, but only twice in
40 WT seedlings analysed). No defect in blade expansion was
observed (Fig. 1J), but cotyledons were frequently epinastically curled
(Fig. 1G).
Leaf blade curling was described in CYCLIN D3;1 (CYCD3;1)overexpressing plants, where it was associated with dramatic
changes in ploidy distribution (Scofield et al., 2013). However,
no significant differences in ploidy distribution were detected
between WT and dek1-4 cotyledons (Fig. 1K). Consistent with this,
no differences in the expression of CYCD3;1, or other cell cyclerelated genes such as LOSS OF GIANT CELLS FROM ORGANS
(LGO), SIAMESE (SIM), KIP-RELATED PROTEIN 6 (KRP6),
HOBBIT (HBT) or DEL1, which encodes an endocycle inhibiting
E2F-like protein, were observed between Col-0 and dek1-4
RESEARCH REPORT
Development (2015) 142, 1978-1983 doi:10.1242/dev.122325
the severity of PC phenotypes (Fig. 3B) and trichome branching
defects (Wilcoxon rank sum test: P=4.24–23 for line C, P=3.03–23 for
line L) (Fig. 3C). Aborted trichomes were observed (Fig. 3D) but not
included in the statistical analysis.
To study the effects of overexpressing the active form of DEK1
(the CALPAIN domain) on the expression of HD-ZIP IV genes, we
used transgenic seedlings harbouring a pRPS5A:CALPAIN-6XHIS
construct (OEX [WT]). Endogenous levels of DEK1 were similar to
that of WT plants, but CALPAIN transcript levels (transgene+
endogenous) were several times higher than in Col-0 seedlings
(supplementary material Fig. S5). No consistent changes in the
expression of HD-ZIP IV genes were observed in these lines
compared with WT seedlings (supplementary material Fig. S5),
suggesting that HD-ZIP IV gene regulation by DEK1 is either
indirect or requires additional factors.
Fine-tuned silencing of DEK1 expression leads to the
appearance of dividing GC-like structures in sepals
Two independent homozygous lines in which amiRDEK1 expression
was triggered only upon DEX application were selected. Induction of
amiRDEK1 expression lowered DEK1 mRNA levels to about half
those detected in control plants (supplementary material Fig. S4J).
Upon flowering, control sepals showed a classic epidermal structure
with GCs surrounded by smaller cells (Fig. 4A). In DEX-grown
plants, we observed sepals with few or no GCs, as well as GC-like
structures that had undergone late divisions after the onset of
differentiation (Fig. 4B-D). Thus, reduced levels of DEK1 affect the
maintenance of GC differentiation.
Cell shape defects in dek1-4 are not caused by changes in
microtubule behaviour
line, but did not exhibit additional defects. In dek1-4 hdg11-2 plants
we observed no significant additivity of the trichome phenotypes
of the two mutants (supplementary material Fig. S2). The sepals of
hdg12-2 dek1-4 and of dek1-4 hdg11-2 mutants resembled those
of dek1-4 mutants (supplementary material Fig. S3). Together, our
results are consistent with multiple HD-ZIP IV proteins acting
downstream of DEK1 to regulate epidermal differentiation.
To confirm whether the phenotype of the dek1-4 allele is caused by
reduced DEK1 activity, we expressed an artificial microRNA (Ossowski
et al., 2008) targeted to the DEK1 coding sequence (amiRDEK1) under
the CaMV 35S promoter and under a dexamethasone (DEX)-inducible
promoter (Craft et al., 2005; Samalova et al., 2005). T1 35S:amiRDEK1
plants showed similar phenotypes to those reported for AtDEK1RNAi
lines (Johnson et al., 2005) and the dek1-4 mutant (Roeder et al., 2012).
Some seedlings had fused cotyledons, discontinuous epidermis and
died. Surviving seedlings had PCs with shallow lobes, true leaves with
multi-branched trichomes, twisted rosette leaves with elongated
petioles and sepals devoid of GCs (supplementary material Fig. S4).
Three independent homozygous lines showing reduced expression
levels of DEK1 (Fig. 3A) were analysed. The expression levels of all
HD-ZIP IV genes previously analysed were not only reduced, but also
correlated tightly with the levels of DEK1 transcript (Fig. 3A) and with
1980
DEK1 activity may define epidermal cell-cell contact zones
A recent study has provided strong evidence that the expression of
HD-ZIP IV-encoding genes is maintained by intercellular signalling
mediated by ACR4 (San-Bento et al., 2014). We measured the
height of epidermal cell contact zones and found that they are less
uniform in height in dek1-4 than in WT seedlings (supplementary
material Fig. S7). Consistent with a defect in epidermal cell
adhesion (Singh et al., 2005), we also observed examples of cell
separation in the cotyledons of 35S:amiRDEK1 lines and the
abnormal accumulation of callose at epidermal cell boundaries in
DEVELOPMENT
Fig. 2. dek1-4 mutants show reduced expression of differentiationpromoting HD-ZIP IV TF-encoding genes. (A,B) Ten-day-old Col-0 (A) and
dek1-4 (B) seedlings. (C) Distribution of branch point numbers in the first and
second true leaves of 15-day-old plants. Number of trichomes: n=500 for Col-0
and n=400 for dek1-4. (D) Gene expression levels in 6-day-old Col-0 and dek1-4
seedlings were quantified by qRT-PCR and normalised using the expression of
EIF4A. Shown is the average of three independent biological replicates±s.d.
PC shape is affected in mutants with altered cortical microtubule
(CMT) dynamics (Ivakov and Persson, 2013). The dek1-4 allele was
introgressed into a line harbouring a GFP-TUA6 expression construct
(Ueda et al., 1999) and the resulting plants analysed. In WT seedlings,
small polygonal PCs have highly oriented CMTs arranged
transversely with respect to their long axis, while in more lobed
PCs CMTs usually orient more randomly and bundle densely in neck
regions. In dek1-4, CMTs are mainly oriented perpendicularly to the
long axis of the cell (supplementary material Fig. S6B,C).
Quantification confirmed that CMTs in mutant PCs are more highly
oriented than in WT PCs (supplementary material Fig. S6D).
To test whether their abnormal arrangement in the mutant was
due to an inability of CMTs to rearrange their distribution, or was a
consequence of cell shape defects, we studied CMT reorganisation
upon ablation in cotyledons (Sampathkumar et al., 2014). CMTs in
both mutant and WT cotyledons reoriented in cell files surrounding
the ablation site (supplementary material Fig. S6E-H), suggesting
that CMT dynamics are not disrupted in dek1-4. This observation
supports the hypothesis that the CMT arrangement in the mutant is a
consequence of altered cell shape.
RESEARCH REPORT
Development (2015) 142, 1978-1983 doi:10.1242/dev.122325
the expanding cotyledons of dek1-4 seedlings (supplementary
material Fig. S7).
Unlike animal calpains, which exist in many isoforms, DEK1 is a
single-copy gene unique to land plants (Liang et al., 2013; Lid et al.,
2002). Here we show that defects in DEK1 perturb epidermal cell
differentiation via a mechanism affecting the transcription of genes
encoding HD-ZIP IV family TFs. The ancestral HD-ZIP IV protein,
like DEK1, is thought to have arisen in the common algal ancestor
of land plants, where it might have been crucial in the evolution of a
specialised outer cell layer with the characteristics necessary for life
on land, such as cuticle formation and stomatal development
(Zalewski et al., 2013). Apoplastic signalling mediated by ACR4,
like DEK1 function, is necessary for the maintenance of HD-ZIP IV
gene expression (San-Bento et al., 2014). However, genetic analysis
carried out in Arabidopsis and maize (Becraft et al., 2002; Roeder
et al., 2012) suggests that DEK1 and ACR4 act in parallel.
A key requirement of the epidermis is that it should be continuous,
implying the maintenance of a highly regulated zone of
circumferential contact between neighbouring cells. A common
feature of phenotypes associated with loss of DEK1 activity is
epidermal cell separation, suggesting that DEK1 might regulate this
zone of cell-cell contact (Ahn et al., 2004; Johnson et al., 2005; Lid
et al., 2005). Here we show that although dek1-4 mutants produce a
continuous epidermis, the zone of cell-cell contact between epidermal
cells is perturbed, further supporting this hypothesis. We propose that
DEK1 influences HD-ZIP IV gene expression, and thus epidermal
differentiation, by regulating epidermal cell adhesion, a function that
might have been crucial in the successful move of plants onto dry land.
1981
DEVELOPMENT
Fig. 3. DEK1 transcript levels correlate with
HD-ZIP IV gene expression and with
epidermal phenotypes. (A) Gene expression
levels in the aerial parts of 6-day-old Col-0 and
35S:amiRDEK1 seedlings were quantified by
qRT-PCR and normalised using the
expression of EIF4A. Shown is the average of
three independent biological replicates±s.d.
(B) Confocal images of the adaxial side of
4-day-old cotyledons stained with PI. Scale
bars: 50 μm. (C) Distribution of branch point
numbers in the first and second true leaves of
15-day-old plants. Number of trichomes:
n=507 for Col-0, n=515 for line E, n=498 for
line C and n=143 for line L. Red bar, aborted
trichomes. (D) Scanning electron micrographs
of true leaves from 15-day-old plants. Scale
bars: 50 μm.
RESEARCH REPORT
Development (2015) 142, 1978-1983 doi:10.1242/dev.122325
For tissue imprints, we used an agarose-based method (Mathur and
Koncz, 1997). Light micrographs were taken with an Axio Imager.M2
microscope (Carl Zeiss) using DIC optics.
RNA extraction and quantitative gene expression analysis
Seedlings were grown vertically for 6 days on square plates and aerial parts
harvested. Three independent biological replicates of 15-20 seedlings were
used. RNA extraction, DNase treatment and qPCR analysis were carried out
as described previously (Xing et al., 2013). Expression levels of each gene,
relative to EIF4A, were determined as previously described (Ferrari et al.,
2006). Primers used for qRT-PCR analysis are shown in supplementary
material Table S1.
Ploidy analysis
Cotyledons were harvested with forceps, chopped with a razor blade in
nuclear lysis buffer (Dewitte et al., 2007), filtered and stained with DAPI.
The nuclei-containing solution was analysed with a Partec ploidy analyser.
Three biological replicates (20 cotyledons from ten independent plants for
each replicate) were performed.
Acknowledgements
Fig. 4. Silencing of the DEK1 gene leads to the appearance of dividing
GCs in sepals. Agarose imprints of sepal epidermis from DEX-grown Col-0 (A)
and amiRDEK1 line 2 (B,C) and 3 (D) plants. GCs are false coloured in pink.
Arrows indicate divisions that have occurred in GCs; dotted lines indicate
common division planes in a group of cells. Scale bar: 50 μm.
We thank Adrienne Roeder (Cornell University, USA) for providing the original dek1-4
mutant line and for helpful discussions; Ian Moore (University of Oxford, UK) for
providing the binary vector pOPON2.1; and Jeremy Just (RDP, ENS de Lyon,
France) for helping with statistical analysis.
Competing interests
The authors declare no competing or financial interests.
MATERIALS AND METHODS
Plant material and growth conditions
Author contributions
Seeds were obtained from the Nottingham Arabidopsis Stock Centre unless
otherwise stated. The dek1-4 allele was donated by Adrienne Roeder
(Cornell University, USA) and introgressed into the Col-0 background.
dek1-4 genotyping involved amplifying genomic DNA using the primers
shown in supplementary material Table S1 and sequencing the resulting
product with primer RG15 (supplementary material Table S1, Fig. S2).
T-DNA insertion lines were genotyped using the primers described in
supplementary material Table S1. A DEK1-specific artificial microRNA
was designed using MicroRNA Designer (WMD, http://wmd3.weigelworld.
org) and generated using the PCR primers in supplementary material
Table S1 according to the protocol from WMD. The pre-amiRNA was
recombined into pDONR221 (Invitrogen) and then into the Gatewaycompatible binary vector pART27 (Lee and Gelvin, 2008) under control of a
CaMV 35S promoter, or into the pOPON2.1 vector (a gift from Ian Moore,
University of Oxford, UK). Plants expressing pRPS5A:CALPAIN-6XHIS
were generated as described for pRPS5A:CALPAIN-GFP (Johnson et al.,
2008), but with the insertion of an in-frame two times 6×His tag with a
STOP codon in place of the GFP sequence.
Soil-grown plants were placed at 20°C and 55% relative humidity under a
16-h light/8-h dark cycle. For in vitro cultures, seeds were surface sterilised with
chlorine gas, stratified on media for 2-3 days in the dark and germinated under a
16-h light/8-h dark cycle at 21°C. To induce DEK1 silencing, germinated
seedlings were transferred after 5 days onto medium supplemented with 0.4%
DMSO (as control) or 25 µm dexamethasone (DEX).
R.G. and G.C.I. designed the experiments and wrote the paper; R.G., G.C.I.,
S.S. and A.M.W. performed the experiments; K.L.J. and R.S.-B. generated materials;
J.A.H.M. contributed analytic tools.
Scanning electron microscopy samples were mounted on graphite paste
(Electron Microscopy Sciences) and visualised using an SH-3000 table-top
scanning electron microscope (HIROX) at −30°C or −50°C with an
accelerating voltage of 5 kV.
Cotyledon PC cell walls were stained with 1 mg/ml propidium iodide (PI)
for 5 min, briefly rinsed, and imaged at a point midway between the midrib
and margin and halfway along the blade. Cell areas and perimeters were
measured using ImageJ (NIH). A ‘shape factor’ (SF=4π area/perimeter2)
was calculated for each cell (Dewitte et al., 2007). CMT alignments were
quantified using the ImageJ plug-in FibrilTool (Boudaoud et al., 2014). All
confocal images were acquired using an LSM700 confocal microscope
(Carl Zeiss).
1982
R.G. was supported by an Agence Nationale de la Recherche (France) Chaire
D’ Excellence [ANR-10-CHEX-0011 - 01 to G.C.I.], R.S.-B. by the Marie Curie ITN
SIREN, and A.M.W. by a Victorian Government postdoctoral training fellowship.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.122325/-/DC1
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